Prognostic 7-SLC-Gene Signature Identified via Weighted Gene Co-Expression Network Analysis for Patients with Hepatocellular Carcinoma

Background Solute carrier (SLC) proteins play an important role in tumor metabolism. But SLC-associated genes' prognostic significance in hepatocellular carcinoma (HCC) remained elusive. We identified SLC-related factors and developed an SLC-related classifier to predict and improve HCC prognosis and treatment. Methods From the TCGA database, corresponding clinical data and mRNA expression profiles of 371 HCC patients were acquired, and those of 231 tumor samples were derived from the ICGC database. Genes associated with clinical features were filtered using weighted gene correlation network analysis (WGCNA). Next, univariate LASSO Cox regression studies developed SLC risk profiles, with the ICGC cohort data being used in validation. Result Univariate Cox regression analysis revealed that 31 SLC genes (P < 0.05) were related to HCC prognosis. 7 (SLC22A25, SLC2A2, SLC41A3, SLC44A1, SLC48A1, SLC4A2, and SLC9A3R1) of these genes were applied in developing a SLC gene prognosis model. Samples were classified into the low-andhigh-risk groups by the prognostic signature, with those in the high-risk group showing a significantly worse prognosis (P < 0.001 in the TCGA cohort and P=0.0068 in the ICGC cohort). ROC analysis validated the signature's prediction power. In addition, functional analyses showed enrichment of immune-related pathways and different immune status between the two risk groups. Conclusion The 7-SLC-gene prognostic signature established in this study helped predict the prognosis, and was also correlated with the tumor immune status and infiltration of different immune cells in the tumor microenvironment. The current findings may provide important clinical indications for proposing a novel combination therapy consists of targeted anti-SLC therapy and immunotherapy for HCC patients.


Introduction
Liver cancer ranks as the second highest cause of tumorresulted mortality [1]. Hepatocellular carcinoma (HCC) constitutes 90% of liver cancer cases. Despite signifcant advances in therapeutic approaches, the recurrence, progression, and metastasis rates of HCC remain high, leading to a poor HCC prognosis [2]. At present, the main treatment options available for HCC are systemic transplantation, drug therapy, transcatheter arterial chemoembolization and radiotherapy, ablative therapy, and surgical resection [3]. However, a great number of HCC patients are already at an advanced stage by the time of diagnosis. Due to the complex molecular mechanisms and cellular heterogeneity of HCC, traditional clinical indicators such as AFP, TNM staging, and vascular invasion have limited ability for predicting the prognosis of HCC. Terefore, for facilitating early detection, predicting the prognosis, and guiding individualized treatment, novel, and more accurate methods are required to understand more clearly HCC developmental mechanisms [4].
After G-protein-coupled receptors, the solute carrier (SLC) superfamily encodes the second largest membrane transporter protein and consists of 65 families and approximately 400 SLC transporter proteins that mainly maintain the stability of the intracellular environment through facilitating various soluble molecular substrates exchange across the lipid membrane [5]. Approximately 80% of small chemical molecules are functionally SLC proteins-dependent [6]. SLC proteins participate in various diseases, for instance, cardiovascular diseases, mental disorders, cancers, and some chronic diseases [7]. SLC proteins play diferent roles in tumor development via regulation of biological processes such as chemoresistance, angiogenesis, proliferation, EMT, metastasis, migration, and immunosuppression as well as the regulation of regulating diferent GFS, metalloproteinases (MMPs), TF, signaling cascades, and cytokines [8]. However, the role and signifcance of the SLC family in HCC was not completely clear. How genes are associated in diferent modules and clinical phenotypes could be systematically described by Weighed gene expression network analysis (WGCNA) [9]. Clinical data information of HCC patients with as well as their mRNA expression profles were obtained publicly from databases. Subsequently, WGCNA was performed using data from the TCGA training cohort to screen module genes associated with tumor staging, and analysis of univariate and LASSO Cox data have both shown that SLC22A25, SLC2A2, SLC41A3, SLC44A1, SLC48A1, SLC4A2, and SLC9A3R1 were prognostic SLC markers, which were validated using data from the ICGC. To assess the underlying mechanisms of these genes, we then performed a functional enrichment analysis.

Co-Expression Network of SLC Family Genes.
Previously, using the human gene database GeneCards (https://www.genecards.org/), SLC genes have been identifed [10], and a co-expression network targeting the SLC family was constructed using the WGCNA R package (version 1.68) [9]. Initially, 397 genes of the SLC family in the TCGA-LIHC cohort were selected as input genes for network construction, and between a gene pair, the Pearson correlation similarity matrix was determined and increased to a soft threshold according to the scale-free topological network criteria. Following this, clustering of the adjacency matrix was carried out with topological overlap (1-TOM) plus dissimilarity . Furthermore, to identify gene modules on the dendrogram, a dynamic tree-cutting algorithm was introduced, with 30 being set as the minimum gene number in each module. In each module, module eigengene (ME) refers to the main component in the gene expression. A Pearson correlation was evaluated between MEs and clinical features (tumor stage, tumor grade, and AFP), and the most relevant modules were selected.

Developing and Validating a Gene Model with the SLC Family.
Genes not included in the ICGC database were excluded from the modules. Our results from the univariate Cox regression study suggested a relation of SLC genes to a prognostic efect on overall survival; LASSO-Cox regression analysis was conducted subsequently for genes with P values of <0.05 via the glmnet R package [11] to avoid overftting. Te risk score of HCC patients was evaluated using the SLC risk score � Ʃ(β i * Exp i ), β i is the LASSO coefcient of the gene, whereas Exp i is the level of expression of a gene. Using the median risk score, training cohort patients were classifed into groups of low-risk and high-risk. Subsequently, the diference in OS of the two groups was estimated based on Kaplan-Meier and ROC curves. Subsequently, the validation of the SLC risk model in the ICGC cohort was operated.

Genomics and Genome Studies Using KEGG and GO.
To determine biological functions (which include cellular components [CCs], molecular functions [MFs], and biological processes [BPs]) and pathways (P values of <0.05 indicated signifcant enrichment), we analyzed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses on genes identifed using univariate Cox regression analysis in clusterProfler R package (version 3.14.3) [12].

Analysis of scRNA-seq Data.
ScRNA-seq analysis was carried out in the "Seurat" R package [13]. Te study included at least 10,000 samples containing detected genes. As part of quality control (QC), the following criteria were introduced: (1) excluding genes detected in fewer than fve cells; (2) excluding cells detected fewer than 200 genes overall. By normalizing the merged data frst and then identifying variable features with the FindVariableFeature function, we collected the frst 2000 highly variable genes (based on variance stabilization transformation). We also used the scale data function to scale all the genes, and principal meta-analysis with RunPCA function to reduce the dimensionality of the frst 2000 highly variable genes screened. To fnd cell clusters, we chose DIM � 1 : 15 and used the functions "FindNeighbors" and "FindCluster" (resolution � 0.5). Next, DIM � 1 : 15 was selected and 2 Journal of Oncology further downscaled using UMAP. Ten, the FindAllMarkers function with logFC � 0.25 (diference ploidy) and minpct � 0.25 (expression ratio of minimum diference genes) was used to screen marker genes in 34 subgroups. In the fnal step, an adjusted P < 0.05 was used for screening marker genes. In addition to cluster classifcation, we identifed and annotated the diferent cell clusters via "Celldex" and "Singler" packages in R. Ten, the Monocle package [14] analyzed single-cell trajectory data to discover cell-state transitions and their relationship to the seven SLC genes.  [17]) and the marker gene expression data of each sample were extracted from the dataset. Te samples collected from primary solid tumors, primary tumors, and primary blood-derived cancer (bone marrow or peripheral blood) were screened. All healthy samples were refned, and their expression values were log2 (x + 0.001)-transformed. In addition, the Pearson correlation coefcients were calculated.

Somatic Alteration Data Collection and Analyses.
Somatic alteration data of the TCGA training cohort were extracted from the Genomic Data Commons data portal (https://gdc.cancer.gov/about-data/publications/mc3-2017) [18], and the maftools R package [19] was used to identify and visualize low-risk and high-risk SLC mutations in the top 20 highest mutation frequencies.

Identifcation of SLC Family Genes Associated with the
Prognosis of HCC. Figure 1 shows the study fow chart. Ultimately, we included 231 patients with HCC from the ICGC (LIRI-JP) cohort and 365 patients with HCC from the TCGA-LIHC cohort. We identifed 397 well-defned SLC genes, and their expression data were taken from the TCGA-LIHC dataset.

Co-Expression Network of SLC and Clinical Features.
WGCNA was performed using data from the TCGA-LIHC cohort. Tree co-expression models were clustered in the hierarchical clustering tree (Figure 2(a)). According to the MEturquoise module, relatively strong positive correlations with tumor stage (Cor � 0.28, P � 4e − 7) and grade (Cor � 0.31, P � 3e − 8) were found (Figures 2(b) and 2(c)). A total of 105 genes were included in the MEturquoise module. As shown in Figure 2(d), 90.5% (95/105) genes in the MEturquoise module were co-expressed in the ICGC (LIRI-JP) dataset and were subsequently subjected to univariate Cox regression analysis. Tirty one prognosis-associated genes were identifed (P value <0.05) (Figure 2(e)). Next, these 31 genes were subjected to GO and KEGG analyses. Organic anion transport was the main enriched BP term, whereas parietal plasma membrane and anion transmembrane transport protein activity were the main enriched CC and MF terms, respectively ( Figure 2(f )). GABAergic synapses, central carbon metabolism in cancer, and bile secretion were signifcantly enriched KEGG pathways ( Figure 2(g)).

Seven SLC Genes Were Verifed in HCC by scRNA-seq
Analysis. Te dataset GSE149614 consists of 71,915 single cells that were then subjected to scRNA-seq analysis, and unsupervised classifcation was successful in classifying the cells into 34 clusters (Figure3(a)). Tese 34 cell clusters showed diferent expression patterns (Figure 3(c)). Our analysis of CellMarker markers determined the nine cell types using "celldex" and "SingleR" markers, namely, 1) hepatocytes; 2) B_cell; 3) endothelial_cell. 3) endothelial_cells; 4) iPS_cells; 5) macrophage; 6) monocyte; 7) NK_cell; 8) smooth_muscle_cells; 9) T_cells ( Figure 3(b)). Moreover, we evaluated the diferential expression characteristics of the nine cell types (Figure 3(d)) and identifed the expression of 7 SLC genes in the nine cells (Figures 3(e), 4(a)-4(o)). We found that the expression of 7 SLC genes was higher in hepatocytes and iPS_cells. Tis may be related to the involvement of SLCs in the conversion of CSCs to HCC [21]. Te risk scores were signifcantly higher in patients who were deceased than in those who survived (P value <0.001) ( Figure 5(d)). Te heat map in Figure 5(e) shows a comparison of tumor and normal tissues in the expression levels of the 7 SLC-related genes. Patients in the training cohort were categorized into low-and high-risk groups by their median risk score ( Figure 6(a)), and a higher risk score meant a greater likelihood of a shorter survival or death (Figures 6(b) and 6(c)) demonstrated seven SLC genes' expression profles in the two risk groups. Principal component analysis showed a bidirectional distribution of patients in the diferent risk groups ( Figure 6(d)). Te predictive performance of the risk score was assessed based on time-dependent ROC curves, with AUC values of 0.75, 0.67, and 0.68 for 1, 2, and 3 years, respectively (Figure 6(e)), indicating that patients with HCC were accurately predicted to survive by the SLC-gene-based signature. A prognostic nomogram was additionally developed (Figure 6(f )), and calibration curves demonstrated that the prediction of 1-and3-year OS was similar to the ideal curve, indicating that patients with HCC were accurately predicted by the nomogram (Figures 6(g) and 6(h)).   Journal of Oncology

Verifcation of the 7-Gene Signature in the ICGC Cohort.
Te accuracy of the constructed risk signature was validated using data from the ICGC dataset though dividing patients into the groups at high-or low-risk as outlined above (Figure 7(a)). We found similar dot plots and heat maps to those in the TCGA cohort (

Association of the Risk Signature with Clinical
Characteristics. Considering the diferent clinical characteristics associated with prognosis in the two risk groups, we investigated the predictive ability of HCC-independent prognostic factors and the risk signature ( Figure 8(c)). In addition, whether the clinical characteristics of HCC were associated with the risk signature was explored. TNM stage and tumor grade were both higher in the high-risk group (Figures 8(d)-8(f)). Te current data indicated that the risk signature could be either used in combination with the clinical indicators available at present or serve as an independent prognostic factor.

GSEA for the Seven-Gene
Signature. Te GSEA technique was applied to the high-risk and low-risk groups of the TCGA training set for studying the functional enrichment of SLC genes. Te R package "limma" detected 12,363 diferentially expressed genes (DEGs) in two risk groups (Figure 9(a)). Te KEGG pathways were identifed by GSEA, and complement and coagulation cascades, retinol metabolism, chemical carcinogenesis, and cytochrome P450 pathways were found to be signifcantly enriched (Figures 9(b) and 9(c)). On verifying these results in the ICGC cohort, 13,071 DEGs were identifed (Figure 10(a)), and bile secretion, chemical carcinogenesis, complement and coagulation cascades, and cytochrome P450 pathways were signifcantly enriched (Figures 10(b) and 10(c)).

Te Relation of SLC-Gene-Based Signature to the Tumor Immune Microenvironment and Immune Cell Infltration.
Te ESTIMATE algorithm was used to calculate the proportion of 28 infltrating immune cells in diferent risk groups to examine the association between the SLCgene-based risk signature and the immune microenvironment. In ICGC and TCGA cohorts, the proportion of activated CD4 T cells, central memory CD4 T cells, regulatory T cells, myeloid-derived suppressor cells, natural killer T cells, and activated dendritic cells were higher in the high-risk group, and that of efector memory CD8 T cells, activated B cells, memory B cells, natural killer cells, eosinophils, and neutrophils was lower in the high-risk group than in the low-risk group (Figures 9(d), 9(f ) and 10(d), 10(e)). In addition, the relationship between SLC-related genes and immune checkpoint genes in patients with HCC was examined using TCGA pan-cancer data ( Figure 11).

Prediction of Chemotherapy Treatment Response in Diferent Risk
Groups. For patients with advanced liver cancer, chemotherapy is a standard treatment. We analyzed the efects of 24 chemotherapeutic agents on HCC in the GDSC database based on the drug "pRRophetic" software package to predict the IC50 of chemotherapeutic agents in HCC patients from diferent SLC risk score groups in the training and testing cohorts. Lower IC50 indicated higher sensitivity to chemotherapeutic drugs. In both TCGA and ICGC cohorts, a higher sensitivity of the high-risk group to sunitinib, cyclopamine, VX-680, imatinib, S-trityl-L-cysteine, Z-LLNIe-CHO, GNF-2, and CGP-082996 was observed, and WZ-1-84 than low-risk group (P < 0.05, Figures 12(a)  and 12(b)).

Multidimensional Validation of the Key Genes in the HPA Database.
To determine the protein expression of the 7 SLC genes, using the HPA database, images of IHC were analyzed. We found that SLC22A25 and SLC2A2 were intensely stained in normal tissues, whereas SLC44A1, SLC9A3R1, SLC48A1, SLC41A3, and SLC4A2 were deeply stained in HCC tissues ( Figure 13). Tese results suggested that these seven genes were specifc markers for SLC.

Discussion
HCC, a polygenic disease, is a complex, multistep process, and the late diagnosis of HCC is a major cause of poor prognosis. Developing new tools for diagnosing HCC can improve its prognosis. High-throughput sequencing facilitates precise treatment, and its use to mine genetic features to predict the prognosis of HCC has become a focus of research. According to a previous study, Zhang et al. constructed a 10-immune-related-lncRNA model using the TCGA and GSE76427 datasets [22]. Li et al. established a 6-gene model related to energy and amino acid metabolism through analyzing TCGA, GSE76427, and ICGC datasets [23]. Liao et al. applied the TCGA database and constructed a 4-gene model based on methylation-related diferentially expressed lncRNAs (MDEs) [24]. Wu et al. analyzed TCGA data, 37 HCC tissues from patients in the Shandong Provincial Hospital, and 11 healthy liver tissues collected from surgically treated patients with liver trauma, and they developed a 4-gene model based on autophagy-related lncRNAs [25]. Song and Chu used the GSE16757, GSE14520, and ICGC datasets and built a 4-gene model based on autophagy-related lncRNAs [26]. Jiang et al. developed a hypoxia-related10-gene model using the TCGA, GSE14520, and ICGC datasets [27]. Despite relatively limited studies of SLC proteins in recent decades, the SLC superfamily is now known to be involved in tumourigenesis, including apoptosis, invasion, proliferation, metastasis, chemoresistance, and other cancer-related processes. Overexpression or suppression of SLC may ofer novel strategies for diagnosis, treatment, or prognosis [28]. Two TS-SLC genes, SLC29A1 (ENT1) and SLC8A1 (NCX1), are downregulated in tumor cells (TCS) via the EMT-induced zinc fnger E box binding homology box 2 (ZEB2)/transforming growth factor (TGF)-BR/nuclear factor (NF)-kB pathway, or miR-223 in HCC, respectively [29]. As a result of enhanced amino acid uptake by SLC38A1 and SLC7A5 (LAT1), and in HCC and TCS grows faster due to YAP/ TAZ pathway activation [30]. Te association between metal ion-mediated tumorigenesis and regulation of various metal transport proteins, including DMT1 (SLC11A2) for iron transport in HCC has been found [31]. Te SLC13A5 gene encodes NaCT, which is seen as a sodium-coupled citrate transporter. NaCT plays a role in fatty acid synthesis, cellular glycolysis, gluconeogenesis cholesterol synthesis, and mitochondrial energy production in the liver [32]. A previous study observed that in liver samples from patients with obesity with insulin resistance and NAFLD, the mRNA expression of SLC13A5 was signifcantly increased, and was correlated with hepatic steatosis [33]. At the inner mitochondrial membrane, the SLC25A13 gene encodes aspartateglutamate carrier 2 (AGC2) to facilitate the calcium-  dependent exchange of cytoplasmic glutamate with mitochondrial aspartate. Te SLC25A13 mutation could not be compensated by other transporter systems in the liver, which would also lead to HCC [34]. In HCC and SLC1A5 directly regulates the mTOR pathway, subsequent growth of HCC cells, and survival signals [35]. Tus, these studies suggested that SLC plays a role in the development and progression of HCC. In this study, the SLC family genes were comprehensively analyzed in HCC and SLC genes associated with the clinical features of HCC were identifed via WGCNA. In addition, a 7gene prognostic model of SLC (SLC22A25, SLC2A2, SLC41A3, SLC44A1, SLC48A1, SLC4A2, and SLC9A3R1; univariate Cox and LASSO regression algorithms) was designed and validated. Te overall survival of training and validation cohort patients was consistently lower in the high-risk group, suggesting that the SLC-based signature assessment of HCC prognosis was accurate and generalizable. Furthermore, ROC analysis was performed to validate the sensitivity and specifcity of the prognostic signature.SLC2A2 encodes glucose transporter protein 2 (GLUT2), which is associated with glycolysis and gluconeogenesis in the liver via the HNF4a-GLUT2 pathway that can afect the uptake and utilization of glucose by HCC cells and is involved in the systemic metabolism of cancer cachexia [36,37]. Te SLC4A2 gene encodes bicarbonate-chloride anion exchange protein 2 (AE2), which mediates proton leakage across the Golgi membrane and allows the Golgi apparatus to act as a proton reservoir in cancer cells, thereby regulating the pH microenvironment of TCS and promoting tumourigenesis and progression [38]. Malfunction of the acid-base homeostasis caused by SLC4A2 can also afect mitochondrial gradients and trigger ROS damage, leading to apoptosis, proliferation, and morphological alterations [39]. SLC9A3R1 encodes the sodium-hydrogen exchange regulator protein (NHERF1) and directly interacts with the PTEN pathway, and its deletion results in increased cell proliferation and Akt activation. Terefore, NHERF1 plays a tumorsuppressive role [40]. NHERF1 regulates Wnt signaling through maintaining a low level of β-catenin protein activation [41]. SLC9A3R1 regulates cancer cell proliferation and metastasis by enhancing PTEN levels to stimulate autophagy, subsequently inhibiting the PI3K-AKT1-MTOR pathway [42]. Te SLC22 family proteins are known as "drug" transporters. Tis family of organic ion transporters mediated the excretion of drugs, endogenous substances, and environmental toxins in vivo, including the subgroups of OATs, OCTs, and OCTNs. Te OATS4 member SLC22A25 is associated only with bound hormones, making it a relatively single specifc transporter protein [43]. SLC22A25 is found in the liver, wherein high co-localization of glucuronide and sulfate is found with androgens and other gonadal steroids [44]. SLC41A3 encodes a mitochondrial Na + -dependent Mg 2+ efux system that regulates the intracellular Mg 2+ homeostasis [45]. Mg 2+ binds to various proteins and is involved in various cellular functions, including genome stabilization and immune responses [46]. Aberrant Mg 2+ levels in cancers have been detected and this could promote cancer progression [47]. In several GEO (GSE36376, GSE22058, GSE64041, GSE76427, GSE63898, GSE14520, GSE54236) and ICGC (ICGC-LIRI) datasets, an increase in SLC41A3 level was found in tumor tissues compared to healthy tissues. A study by Liu et al. demonstrated that HCC patients with low levels of SLC41A3 expression have signifcantly better outcomes (OS). Compared to healthy tissues, LIHC had a signifcantly lower DNA methylation level of SLC41A3, which may account mainly for a high-expressed SLC41A3 in tumor tissues [48]. SLC44A1 encodes choline transporter-like protein 1 (CTL1) and is found in both plasma and mitochondrial membranes. SLC44A1 transports choline in a sodium-ion-nondependentmoderate-afnity manner [49]. CTL1- Journal of Oncology 13   1100  1200  1300  1400  1500  1600  1700  1800  1900  2000  2100  2200  2300  2400  2500  0  100  200  300  400  500  600  700  800  900  1000  2600  2700  2800  2900  3000  3100  3200  3300  3400  3500  3600  3700  3800  3900        mediated choline transport is a critical step in synthesizing phospholipids that form a plasma membrane. Apoptosis can be induced by inhibiting choline uptake [50]. Cancer cells have enhanced choline uptake via CTL1, which promotes membrane phospholipid synthesis and cell proliferation. Terefore, CTL1 could be a new target molecule for cancer therapy [51]. SLC48A1 is a heme transporter mainly found in endosomes and is involved in the transport of heme iron during iron metabolism. It encodes a facilitator transporter protein (HRG-1), which regulates the V-ATPase activity, enhances glucose transporter-1 (GLUT-1) transport, increases glucose uptake and lactate production, and promotes insulin-like growth factor I receptor (IGF-1R) transport [52]. Furthermore, overexpression of SLC48A1 promotes invasion, migration, and glycolysis, and cancer   ADORA2A  ARG1  BTLA  CD274  CD276  CTLA4  EDNRB  HAVCR2  IDO1  IL10  IL13  IL4  KIR2DL1  KIR2DL3  LAG3  PDCD1  SLAMF7  TGFB1  TIGIT  VEGFA  VEGFB  C10orf54  VTCN1  GZMA  BTN3A1  BTN3A2  CCL5  CD27  CD28  CD40  CD40LG  CD70  CD80  CX3CL1  CXCL10  CXCL9  ENTPD1  HMGB1  ICAM1  ICOS  ICOSLG  IFNA1  IFNA2  IFNG  IL1A  IL1B  IL2  IL2RA  ITGB2  PRF1  SELP  TLR4  TNF  TNFRSF14  TNFRSF18  TNFRSF4  TNFRSF9  TNFSF4  TNFSF9  IL12A   SLC22A25  SLC2A2  SLC41A3  SLC44A1  SLC48A1  SLC4A2 Figure 11: Correlations between SLC-related gene signature and immune checkpoint genes are mapped out as a heatmap. Red represents positive correlation, and blue represents negative correlation; the darker the color, the better the correlation.
cell growth, which are associated with less favorable outcomes [53]. Still, this study had certain limitations. We utilized a public database to conduct a retrospective bioinformatics analysis, but it would be more convincing if the SLC-gene-based risk signature was cross-validated in more samples. In addition, the specifc biological role of the seven prognostic SLC genes in HCC should be validated via molecular and animal experiments.

Conclusion
In conclusion, the 7-gene signature based on SLC genes showed a satisfactory accuracy and generalizability in predicting the survival outcomes of patients with HCC. In addition, in the tumor microenvironment, the signature was related to the tumor immune status and infltration of diferent immune cells. Terefore, this study provided novel insights into developing SLC-based treatment strategies for HCC.

Ethical Approval
As the data are freely available and our study is retrospective, ethical approval, and informed consent are dispensed.

Conflicts of Interest
Te authors declare that they have no conficts of interest.